PCT/EP O 3 I O 8 6 6 6
`
`REC'D 2 0 OCT 2003
`
`WIPO
`
`PCT
`
`UNITED STA~ DEPARTMENT OF COMMERCE
`
`United States Patent and Trademark Office
`
`September 04, 2003
`
`TIDS IS TO CERTIFY THAT ANNEXED HERETO IS A TRUE COPY FROM
`THE RECORDS OF THE UNITED STATES PATENT AND TRADEMARK
`OFFICE OF THOSE PAPERS OF THE BELOW IDENTIFIED PATENT
`APPLICATION THAT MET THE REQUIREMENTS TO BE GRANTED A
`FILING DATE UNDER 35 USC 111.
`
`APPLICATION NUMBER: 60/402,541
`FILING DA TE: August 12, 2002
`
`I
`. PRIORITY DOCUMENT
`SUBMITTED OR TRANSMITTED IN
`COMPLIANCE WITH
`RULE 17.l(a) OR (b)
`
`T. LAWRENCE
`Certifying Officer
`
`Alnylam Exh. 1007
`
`i
`
`

`

`60'-!,,Q:::it.1!:j;~.
`Attorney Docket Number.
`
`•· -
`
`.~
`
`•
`
`----- 'tt
`·.;
`0
`
`Please type a plus sign (+) mide 1h15 box (cid:157) r:;7
`·-
`·• c..,
`~
`=-= .. o . .
`PROVISIONAL APPLICATION FOR PATENT COVER SHEET g =-
`~ _ ~ -
`:;;~ =~
`~ SC7 C: Box PROVISIONAL PATENT APPLICATION
`~ ; ~c.
`t-.>:::·
`~n--... oo
`fl>~ =!;:
`COMMISSIONER FOR PATENTS
`WASHINGTON, D.C. 20231
`- - -~
`THIS IS A REQUEST FOR FILING A PROVISIONAL APPLICATION FOR PATENT UNDER 37 C.F.R. § 1.53(C). ~c:::> i:;;'j
`... 11
`C
`.....
`INVENTOR(S)/APPLICANT(S)
`Residence (City and Either State or Foreign Country)
`Family Name or sumame
`KAUFMANN
`Berlin, Germany
`GIESE
`Berlin, Germany
`
`Given Name (firsl and m!ddle (if any))
`Jorg
`Klaus
`
`Additional inventors are being named on page 2 attached hereto.
`TITLE OF THE INVENTION (280 characters max)
`Novel Forms of Interfering RNA Molecules
`CORRESPONDENCE ADDRESS
`
`(cid:143)
`
`Please Direct All Correspondence To:
`~ Customer No.
`30082
`(cid:143)
`Firm Name
`Attorney of Record
`Address
`
`City
`Country
`
`~ Specification
`
`Number of Pages
`
`Heller Ehrman White & McAuliffe LLP
`Paul M. Booth
`Intellectual Property Department
`101 Orchard Ridge Drive
`Suite JOO
`I Zip Code I 20878-1917
`I
`Gaithersburg
`State
`IMO
`I Facsimile I 301-721-6299
`I Telephone I 301-121-6100
`U.S.A.
`ENCLOSED APPLICATION PARTS (check all that apply)
`
`49 ~ Small Entity Status Claimed As:
`0 Independent Inventor
`181 Small Business Concern
`0 Non-Profit Organization
`
`~ Drawlng(s)
`
`19
`METHOD OF PAYMENT OF FILING FEE FOR THIS PROVISIONAL APPLICATION
`
`Number of Sheets
`
`(cid:143) Other (specify)
`(cid:143)
`[Z} The Commissioner is hereby authorized to charge the $ 80.00 filing fee to Deposit Account No. 08-
`
`A check in the amount of$ 80.00
`
`is enclosed to cover the filing fee.
`
`1641. In the event any variance exists, please charge or credit any such variance to Deposit Account
`No. 08-1641.
`The invention was made by an agency of the United States Government or under a contract with an agency of
`the United States Government.
`181
`No.
`D
`Yes, the name of the U.S. Government agency and the Government contract number are:
`
`Respecifully ~ ,,,..-
`_,,.--
`.,....,
`' v~·
`Paul M. 8 ootn
`
`By
`
`Date
`Telephone
`Registration No.
`
`August 12, 2002
`301-721-6100
`40,244
`
`)
`
`Paga 1 of 1
`
`ii
`
`

`

`atugen AG
`A 19005 EP
`
`
`
`Novel formsof interfering RNA molecules
`
`
`
`The present invention is related to a rrbonucleic acid comprising a double-stranded structure
`whereby the double-stranded structure comprises a first strand and a second strand, whereby
`the first strand comprises a first stretch of contiguous nucleotides and wherebysaid first
`stretch is at least partially complementary to the target nucleic acid, and the second strand
`comprises a second stretch of contiguous nucleotides whereby said secondstretch is at least
`partially identical to a target nucleic acid, the use of such ribonucleic acid, a cell and an
`organism, respectively, comprising such ribonucleic acid, a composition containing such
`ribonucleic acid, a pharmaceutical composition containing such ribonucleic acid and a method
`
`for inhibiting expression of a targeted gene.
`
`is a post-transcriptional gene silencing mechanism
`RNA-mediated interference (RNAi)
`initiated by double stranded RNA (dsRNA) homologous in sequence to the silenced gene
`(Fire (1999), Trends Genet 15, 358-63, Tuschl, et al.
`(1999), Genes Dev 13, 3191-7,
`,
`Waterhouse, et al. (2001), Nature 411, 834-42, Elbashir, ef al. (2001), Nature 411, 494-8, for
`review see Sharp (2001), Genes Dev 15, 485-90, Barstead (2001), Curr Opin Chem Biol5,
`63-6). RNAi has been used extensively to determine gene function in a number of organisms,
`including plants
`(Baulcombe (1999), Curr Opin Plant Biol 2,
`109-13), nematodes
`(Montgomery, ef al. (1998), Proc Natl Acad Sci U S A 95, 15502-7), Drosophila (Kennerdell,
`et al. (1998), Cell 95, 1017-26, Kennerdell, et al. (2000), Nat Biotechnol 18, 896-8). In the
`nematode C. elegans about one third of the genomehas already been subjected to functional
`analysis by RNAi (Kim (2001), Curr Biol 11, R85-7, Maeda, et al. (2001), Curr Biol 11, 171-
`
`6).
`
`Until recently RNAi in mammalian cells was not generally applicable, with the exception of
`early mouse development (Wianny, et al. (2000), Nat Cell Biol 2, 70-5). The discovery that
`transfection of duplexes of 21-nt into mammalian cells interfered with gene expression and
`did not induce a sequence independentinterferon-driven anti-viral response usually obtained
`with long dsRNAled to new potential application in differentiated mammaliancells (Elbashir
`
`1
`
`

`

`\
`
`iexd*
`anthe
`oy CY aC ES
`
`-7ES eee
`
`interfering RNAs (siRNAs )
`et al. (2001), Nature 411, 494-8). Interestingly these small
`resemble the processing products from long dsRNAs suggesting a potential bypassing
`mechanism in differentiated mammalian cells. The Dicer complex, a member of the RNAse
`
`Ill family, necessary for the initial dsRNA processing has been identified (Bernstein, et al.
`
`(2001), Nature 409, 363-6, Billy, et al. (2001), Proc Natl Acad Sci US A 98, 14428-33). One
`
`of the problems previously encountered when using unmodified nbooligonucleotides was the
`
`rapid degradation in cells or even in the serum-containing medium (Wickstrom (1986), J
`
`Biochem Biophys Methods 13, 97-102, Cazenave, et al. (1987), Nucleic Acids Res 15,
`
`10507-21).
`
`It will depend on the particular gene function and assay systems used whetherthe
`
`respective knock down induced by transfected siRNA will be maintained long enough to
`
`achieve a phenotypic change.
`
`The problem underlying the present
`
`invention was to provide synthetic interfering RNA
`
`molecules which are both stable and active ina biochemical environment such asa livingcell.
`
`invention the problem is solved by a ribonucleic acid
`In a first aspect of the present
`comprising a double stranded structure whereby the double- stranded structure comprises a
`first strand and a second strand, whereby the first strand comprises a first stretch of
`
`contiguous nucleotides and wherebysaid first stretch is at least partially complementary to a
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`nucleotides whereby said secondstretch is at least partially identical to a target nucleic acid,
`
`and whereby the double stranded structure is blunt ended.
`
`In a second aspect the problem underlying the present invention is solved by a ribonucleic
`acid comprising a double stranded structure whereby the double- stranded structure comprises
`a first strand and a second strand, whereby the first strand comprises a first stretch of
`
`contiguous nucleotides and wherebysaidfirst stretch is at least partially complementary to a
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`nucleotides, whereby said secondstretch is at least partially identical to a target nucleic acid,
`wherebythefirst stretch and/or the second stretch have a length of 18 or 19 nucleotides.
`
`In an embodimentof the ribonucleic acid accordingto the first aspect of the invention thefirst
`
`stretch and/or the second stretch have a length of 18 or 19 nucleotides.
`
`2
`
`

`

` ACoeOe ee
`
`In a further embodimentof the ribonucleic acid according to the first aspect of the invention
`
`the double stranded structure is blunt ended on both sides of the double strand.
`
`In an alternative embodiment of the ribonucleic acid according to the first aspect of the
`
`invention the double stranded structure is blunt ended on the double stranded structure which
`
`is defined by the 5’-end of the first strand and the 3’-end of the secondstrand.
`
`In a further alternative embodimentof the ribonucleic acid accordingto the first aspect of the
`
`invention the double stranded structure is blunt ended on the double stranded structure which
`
`is defined by the 3’-endof the first strand and the 5’-end of the secondstrand.
`
`In a third aspect the problem underlying the present invention is solved by a ribonucleic acid
`
`comprising a double stranded structure whereby the double- stranded structure comprises a
`
`first strand and a second strand, whereby the first strand comprises a first stretch of
`
`contiguous nucleotides and wherebysaid first stretch is at least partially complementary to a
`
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`
`nucleotides and whereby said second stretch is at least partially identical to a target nucleic
`
`acid, and wherebyat least one of the two strands has an overhangof at least one nucleotide at
`
`the 5’-end.
`
`In an embodimentof the ribonucleic acid according to the third aspect of the present invention
`
`the overhang consists of at least one nucleotide which is selected from the group comprising
`
`ribonucleotides and desoxyribonucleotides.
`
`In a more preferred embodimentof the ribonucleic acid according to the third aspect of the
`
`present invention the nucleotide has a modification whereby said modification is preferably
`selected from the group comprising nucleotides being an inverted abasic and nucleotides
`
`having an NH2-modification at the 2’-position.
`
`In a preferred embodimentof the ribonucleic acid according to the third aspect of the present
`invention at least onc of the strands has an overhang of at least one nucleotide at the 3’-end
`
`consisting of ribonucleotide or deoxyribonucleotide.
`
`3
`
`

`

`ed bala acaSe
`
`4
`
`In another preferred embodimentof the ribonucleic acid according to the third aspect of the
`
`present
`
`invention the first stretch and/or the second stretch have a length of 18 or 19
`
`nucleotides.
`
`In an embodiment of the ribonucleic acid according to any aspect of the present invention the
`
`double-strandedstructure has a length of 17 to 21 nucleotides, preferably 18 to 19 nucleotides
`
`In an embodimentof the ribonucleic acid according to the third aspect of the present invention
`
`the overhang at the S’-endis on the secondstrand.
`
`In a preferred embodimentof the ribonucleic acid according to the third aspect of the present
`inventionthefirst strand comprises also an overhang, preferably at the 5’-end.
`
`In an embodimentof the ribonucleic acid accordingto the third aspect of the present invention
`
`the 3’-end of the first strand comprises an overhang.
`
`In an alternative embodiment of the ribonucleic acid according to the third aspect of the
`
`present invention the overhang at the 5’-endis on the first strand.
`
`In a preferred embodiment thereof the second strand also comprise an overhang, preferably at
`
`the 5’-end.
`
`In an embodiment ofthe ribonucleic acid according to the third aspect of the present invention
`
`the 3’-end of the first strand comprises an overhang.
`
`In an embodimentof the ribonucleic acid according to any aspect of the present invention at
`
`least one nucleotide of the ribonucleic acid has a modification at the 2’-position and the
`
`modification is preferably selected from the group comprising amino, fluoro, methoxy and
`
`alkyl.
`
`In a fourth aspect the problem underlying the present invention is solved by a ribonucleic acid
`comprising a double stranded structure, whereby the double- stranded structure comprises a
`first strand and a second strand, whereby the first strand comprises a first stretch of
`
`contiguous nucleotides and whereby said first stretch is at least partially complementary to a
`
`4
`
`

`

`
`AW AY GESIaMAO eea
`
`target nucleic acid, and the second strand comprises a second stretch of contiguous
`nucleotides and whereby said secondstretch is at least partially identical to a target nucleic
`
`acid,
`
`whereby
`
`said first strand and/or said second strand comprises a plurality of groups of modified
`
`nucleotides having a modification at the 2’-position whereby within the strand each group of
`
`modified nucleotides is flanked on one or both sides by a flanking group of nucleotides
`
`whereby the flanking nucleotides forming the flanking group of nucleotides is either an
`
`unmodified nucleotide or a nucleotide having a modification different from the modification
`
`of the modified nucleotides.
`
`In an embodiment of the mbonucleic acid according to the fourth aspect of the present
`
`invention the ribonucleic acid is the ribonucleic acid according to the first, second or third
`
`aspect of the present invention.
`
`In a further embodiment of the mbonucleic acid according to the fourth aspect of the present
`
`invention said first strand and/or said second strand comprise said plurality of modified
`
`nucleotides.
`
`In another embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention said first strand comprises said plurality of groups of modified nucleotides.
`
`In yet another embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present
`
`invention said second strand comprises said plurality of groups of modified
`
`nucleotides.
`
`In an preferred embodiment of the ribonucleic acid according to the fourth aspect of the
`present invention the group of nucleotides and/or the group of flanking nucleotides comprises
`
`a number of nucleotides whereby the numberis selected from the group comprising one
`
`nucleotide to 10 nucleotides.
`
`5
`
`

`

`oa
`
`cH
`ers?
`Asa! ims
` ehUNSBs
`
`In another embodimentofthe ribonucleic acid according to the fourth aspect of the present
`invention the pattern of modified nucleotides of said first strand is the same as the pattern of
`modified nucleotides of said second strand.
`
`In a preferred embodimentofthe ribonucleic acid according to the fourth aspect of the present
`invention the pattern of said first strand aligns with the pattern of said sccondstrand.
`
`In an alternative embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present invention the pattern of said first strand is shifted by one or more nucleotides relative
`
`to the pattern of the second strand.
`
`In an embodiment of the ribonucleic acid according to the fourth aspect of the present
`
`invention the modification is selected from the group comprising amino, fluoro, methoxy and
`
`alkyl.
`
`In another embodiment of the mbonucleic acid according to the fourth aspect of the present
`
`invention the double strandedstructure is blunt ended.
`
`In an preferred embodiment of the nbonucleic acid according to the fourth aspect of the
`
`present invention the double strandedstructure is blunt ended on bothsides.
`
`In another embodiment of the mbonucleic acid according to the fourth aspect of the present
`
`invention the double stranded structure is blunt ended on the double stranded structure’s side
`
`which is defined by the 5’-end of the first strand and the 3’-end of the second strand.
`
`In still another embodiment of the ribonucleic acid according to the fourth aspect of the
`
`present
`
`invention the double stranded structure is blunt ended on the double stranded
`
`structure’s side which is defined by at the 3’-end of the first strand and the 5’-end of the
`
`secondstrand.
`
`In another embodimentofthe ribonucleic acid according to the fourth aspect of the present
`
`invention at least one of the two strands has an overhang ofat least one nucleotide at the 5’-
`
`end.
`
`6
`
`

`

`Sut
`
`wuBmp 201
`
`In a preferred embodimentofthe ribonucleic acid according to the fourth aspect of the present
`invention the overhang consists of at least one desox yribonucleotide.
`
`In a further embodimentof the ribonucleic acid according to the fourth aspect of the present
`invention at least one of the strands has an overhangofat least one nucleotide at the 3’-end.
`
`In an embodiment of the ribonucleic acid according to any of the aspects of the present
`
`invention the length of the double-stranded structure has a length from about 17 to 21 and
`
`more preferably 18 or 19 bases
`
`In another embodimentof the ribonucleic acid according to any of the aspects of the present
`invention the length of said first strand and/or
`the length of said second strand is
`
`independently from each other selected from the group comprising the ranges of from about
`
`15 to about 23 bases, 17 to 21 bases and 18 or 19 bases.
`
`In a preferred embodiment of the nbonucleic acid according to any of the aspects of the
`
`present invention the complementarity between said first strand and the target nucleic acid is
`
`perfect.
`
`In an embodiment of the ribonucleic acid according to any of the aspects of the present
`
`invention the duplex formed betweenthe first strand and the target nucleic acid comprises at
`
`least 15 nucleotides wherein there is one mismatch or two mismatches between said first
`
`strand and the target nucleic acid forming said double-stranded structure.
`
`In a preferred embodiment of the ribonucleic acid according to any of the aspects of the
`
`present invention the target gene is selected from the group comprising structural genes,
`housekeeping genes,
`transcription factors, motility factors, cell cycle factors, cell cycle
`
`inhibitors, enzymes, growth factors, cytokines and tumorsuppressors.
`
`In a further embodiment of the ribonucleic acid according to any of the aspects of the present
`
`invention the first strand and the second strandare linked by a loopstructure.
`
`In a preferred embodiment of the ribonucleic acid according to any of the aspects of the
`present invention the loop structure is comprised of a non-nucleic acid polymer.
`
`7
`
`

`

`
`
`ee VY =e
`
`In a preferred embodimentthereof the non-nucleic acid polymeris polyethylene glycol.
`
`In an alternative embodimentthereof the loop structure is comprised of a nucleic acid.
`
`In an embodiment of the ribonucleic acid according to any of the aspects of the present
`invention the 5’-terminusof the first strand is linked to the 3’-terminus of the second strand.
`
`In a further embodimentofthe ribonucleic acid according to any ofthe aspects of the present
`invention the 3’-end ofthefirst strand is linked to the 5’-terminus of the secondstrand.
`
`In a fifth aspect the problem underlying the present invention is solved by the use of a
`ribonucleic acid according to any of the aspects of the present invention, for target validation.
`
`In a sixth aspect the problem underlying the present invention is solved by the use of a
`ribonucleic acid according to any of the aspects of the present invention, for the manufacture
`
`of a medicament.
`
`In a preferred embodimentofthe use according to the sixth aspect of the present invention the
`
`medicament is for the treatment of a disease or of a condition which is selected from the
`
`group comprising glioblastoma, prostate cancer, breast cancer,
`
`lung cancer,
`
`liver cancer,
`
`colon cancer, pancreatic cancer and leukaemia, diabetes, obesity, cardiovascular diseases, and
`
`metabolic diseases.
`
`In a seventh aspect
`
`the problem underlying the present
`
`invention is solved by a cell,
`
`preferably a knockdowncell, containing a ribonucleic acid according to anyof the aspects of
`
`the present invention.
`
`In an eighth aspect the problem underlying the present invention is solved by an organism,
`
`preferably a knockdown organism, containing a ribonucleic acid according to any of the
`
`aspects of the present invention.
`
`In a ninth aspect the problem underlying the present invention is solved by a composition
`
`containing a ribonucleic acid according to any of the aspects of the present invention.
`
`8
`
`

`

`oS ackae a Pee i
`
`In a tenth aspect the problem underlying the present invention is solved by a pharmaceutical
`composition containing a ribonucleic acid according to any of the aspects of th epresent
`invention, and a pharmaceutically acceptable carrier.
`
`In an eleventh aspect the problem underlying the present invention is solved by method for
`inhibiting the expression of a target gene in a cell or derivative thereof comprising
`introduction of a ribonucleic acid according to any of the aspects of the present invention into
`
`the cell in an amount sufficient to inhibit expression of the target gene, wherein the target
`
`gene is the target gene of the a ribonucleic acid according to any of the aspects of the present
`
`invention.
`
`The present invention is based on the surprising finding that small interfering RNAs can be
`
`designed such as to be both highly specific and active as well as stable under the reaction
`
`conditions typically encountered in biological systems such as biochemical assays or cellular
`
`environments. The various interfering RNAsdescribed in the prior art such as by Tuschletal.
`
`(international patent application WO 01/75164) provide for a length of 21 to 23 nucleotides
`
`and a modification at the 3’ end of the double-stranded RNA.It has been surprisingly found
`
`by the present inventors that the problem of stability of interfering RNA, including small
`
`interfering RNA (siRNA) which is generally referred to herein in the following as RNAi,
`
`actually resides in the attack of endonucleases rather than exonucleases as thought earlier.
`
`Based on this finding several strategies have been perceived by the present inventors which
`
`are subject to the present application.
`
`The present invention is thus related to new forms of interfering RNA. RNAi consists of a
`
`ribonucleic acid comprising a double-stranded structure. Said double-stranded structure is
`
`formed by a first strand and a second strand. Said first strand comprises a stretch of
`
`contiguous nucleotides, also referred to as first stretch of contiguous nucleotides herein, and
`
`this first stretch is at least partially complementary to a target nucleic acid. Said second strand
`comprises also a stretch of contiguous nucleotides whereby said second stretch is at least
`partially identical to a target nucleic acid. The very basic structure of this ribonucleic acid is
`
`schematically shown in Fig. 1. Said first strand and said second strand are preferably
`
`hybridised to each other and form the double-stranded structure. The hybridisation typically
`
`occurs by Watson Crick base pairing. The inventive ribonucleic acid, however,
`
`is not
`
`9
`
`

`

`fy Cd SL aL ASSeao
`
`10
`
`necessarily limited in its length to said double-stranded structure. There might be further
`
`nucleotides added to each strand and/or to each end of any of the strands forming the RNAi.
`
`Depending on the particular sequence of the first stretch and the second stretch,
`
`the
`
`hybridisation or base pairing is not necessarily complete or perfect, which meansthat thefirst
`
`and the second stretchare not 100 % base paired due to mismatches. There might also be one
`
`or more mismatches within the duplex. Said mismatches have no effect on the RNAiactivity
`
`if placed outside a stretch of 17 matching nucleotides. If mismatches are placed to yield only
`15 or less contiguous matching nucleotides, the RNAi molecule typically shows a reduced
`activity in down regulating mRNAfor a given target compared to a 17 matching nucleotide
`
`duplex.
`
`The first stretch of contiguous nucleotides is essentially complementary to a target nucleic
`acid, more preferably to a part of the target nucleic acid. Complementary as used herein
`preferably means that the nucleotide sequence ofthe first strand is hybridising to a nucleic
`acid sequenceora part thereofof a target nucleic acid sequence. Typically, the target nucleic
`acid sequence is, in accordance with the mode of action of interfering ribonucleic acids, a
`single stranded RNA, more preferably an mRNA. Such hybridisation occurs most likely
`through Watson Crick base pairing, however, is not necessarily limited thereto. The extent to
`whichsaid first strand and moreparticularly thefirst stretch of contiguous nucleotides of said
`first strand is complementary to a target nucleic acid sequence can be as high as 100% and be
`as little as 80%, preferably 80-100%, more preferably 85-100%, most preferably 90-100%.
`Optimum complementarity seems to be 95-100%. Complementarity in this sense means that
`the aforementioned range of nucleotides, such as, e. g., 80%-100%, depending on the
`particular range, of the nucleotides are perfect by Watson Crick base pairing. It is shown in
`one aspect of the present invention that the complementarity between said first stretch of
`nucleotides and the target RNA has to be 18-19 nucleotides, stretches of as little as 17
`nucleotides even with two sequence specific overhangs are not functional in mediating RNAi.
`Accordingly, given a duplex having a length of 19 nucleotides or base pairs a minimum
`complementarity of 17 nucleotides or nucleotide base pairs would be acceptable allowing for
`a mismatch of two nucleotides. In case of a duplex consisting of 20 nucleotides or base pairs a
`complementarity of 17 nucleotides or nucleotide base pairs would be allowable and
`functionally active. The same applies to a duplex of 21 nucleotides or base pairs with a total
`of 17 complementary nucleotides or base pairs. Basically,
`the extent of complementarity
`required for a length of a duplex, i. e. of a double stranded structure, can also be based on the
`
`10
`
`10
`
`

`

`=
`See hanes!
`22 LEME EMAL AL ER hgee
`
`11
`
`melting temperature of the complex formed by either the double stranded structure as
`described herein or by the complex ofthefirst stretch of the first strand and the target nucleic
`acid.
`
`It is to be understood that all of the ribonucleic acid of the present invention are suitable to
`cause or being involved in RNA mediated interference such as, for example, described in
`internationalpatent applications WO 99/32619, WO 00/44895 and WO 01/75164.
`
`The first strategy according to which an interfering ribonucleic acid molecule may be
`designed according to the present invention is to have an optimum length of 18 or 19
`nucleotides of the stretch which is complementary to the target nucleic acid. It is also within
`the present invention that said optimum length of 18 or 19 nucleotides is the length of the
`double stranded structure in the RNAi used. This length requirementis clearly different from
`the technical teaching ofthe prior art such as, for example, the international patent application
`WO01/75164.It is within the present invention that any further design, both according to the
`present invention and as described in the prior art, can be realised in connection with an
`interfering ribonucleic acid having said length characteristics,
`i.e. a length of 18 or 19
`nucleotides.
`
`The second strategy according to which an interfering ribonucleic acid molecule may be
`designedis to have a free 5° hydroxyl group,also referred to hercin as free 5’ OH-groupat the
`first strand. A free 5° OH-group meansthat the most terminal nucleotide forming the first
`strand is present andis thus not modified, particularly not by an end modification. Typically,
`the terminal 5’-hydroxy group of the second strand, respectively,
`is also present
`in an
`unmodified manner. In a more preferred embodiment, also the 3’-end of the first strand and
`first stretch, respectively, is unmodified such as to present a free OH-group which is also
`referred to herein as free 3’OH-group. Preferably such free OH-groupis also presentat the 3 -
`end of the second strand and second stretch, respectively. In other embodiments of the
`ribonucleic acid molecules according to the present invention the 3’-end ofthe first strand and
`first stretch,
`respectively, and/or the 3’-end of the second strand and second stretch,
`respectively, may have an end modification at the 3’ end.
`
`As used herein the terms free 5°OH-group and 3’OH-group also indicate that the respective
`most terminal nucleotide at thc S’end and the 3° end of the polynucleotide, respectively,
`
`11
`
`11
`
`

`

`CME ES Ne gee ee
`
`12
`
`presents a OH-group. Such OH-group may stem from either the sugar moiety of the
`nucleotide, more preferably from the 5’position in case of the 5°OH-group and from the
`3’position in case of the 3’OH-group, or from a phosphate group attached to the sugar moiety
`of the respective terminal nucleotide. The phosphate group may in principle be attached to
`any OH-group of the sugar moiety of the nucleotide. Preferably, the phosphate group is
`attached to the 5°OH-group of the sugar moiety in case ofthe free 5’QH-group and/or to the
`3°OH-group ofthe sugar moiety in case of the free 3’OH-group.
`
`As used herein with any strategy for the design of RNAi or any embodiment of RNAi
`disclosed herein, the term end modification means a chemical entity added to the most 5’ or 3’
`nucleotide of the first and/or secondstrand. Examples for such end modifications include, but
`are not limited to, inverted (deoxy) abasics, amino, fluoro, chloro, bromo, CN,CF, methoxy,
`imidazole, caboxylate,
`thioate, C;
`to Cio lower alkyl, substituted lower alkyl, alkaryl or
`aralkyl, OCF3, OCN,O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH;; SO.CH;; ONO; NOQz,
`N3; heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino or substituted
`silyl, as, among others, described in European patents EP 0 586 520 BI or EP 0 618 925 Bl.
`
`A further end modification is a biotin group. Such biotin group may preferably be attached to
`either the most 5’ or the most 3’ nucleotide ofthe first and/or second strand or to both ends. In
`
`a more preferred embodiment the biotin group is coupled to a polypeptide or a protein.It is
`also within the scope of the present invention that the polypeptide or protein is attached
`through any of the other aforementioned end modifications. The polypeptide or protein may
`confer further characteristics to the inventive nucleic acid molecules. Among others the
`polypeptide or protein mayact as a ligand to another molecule. If said other molecule is a
`
`receptor the receptor’s function and activity may be activated by the binding ligand. The
`
`receptor may show an internalization activity which allows an effective transfection of the
`
`ligand bound inventive nucleic acid molecules. An example for the ligand to be coupled to the
`inventive nucleic acid molecule is VEGF and the corresponding receptor is the VEGF
`
`receptor.
`
`Various possible embodiments of the RNAiof the present invention having different kinds of
`
`end modification(s) are presented in the following table 1.
`
`12
`
`12
`
`

`

`ats
`2ee ae ie
`
`13
`
`Table 1: Various embodiments of the interfering ribonucleic acid according to the
`present invention
`
`1“ strand/1* stretch
`
`2"!
`
`strand/
`
`2nd
`
`stretch
`
` 1.) 5’-end
` free OH
`37-end
`free OH
`
`
`free OH
`free OL
`
`2.) 5’-end
`3°-end
`
`3.) 5’-end
`3°-end
`
`4.) 5’-end
`3°-end
`
`5.) 5’-end
`3°-end
`
`6.) 5’-end
`3’-end
`
`7.) 5’-end
`3°-end
`
`8.) 5-end
`3’-end
`
`free OH
`end modification
`
`free OH
`end modification
`
`free OH
`free OH
`
`free OH
`end modification
`
`free OH
`end modification
`
`free OH
`free OH
`
`free OH
`free OH
`
`end modification
`free OH
`
`free OH
`end modification
`
`end modification
`free OH
`
`free OH
`free OH
`
`end modification
`end modification
`
`free OH
`end modification
`
`end modification
`end modification
`
`
`
`The various end modifications as disclosed herein are preferably located at the ribose moicty
`of a nucleotide of the ribonucleic acid. More particularly,
`the end modification may be
`attached to or replace any of the OH-groups ofthe ribose moiety, including but not limited to
`the 2’OH, 3’OH and 5’OHposition, provided that the nucleotide thus modified is a terminal
`
`nucleotide. Inverted abasics are nucleotides, either desoxyribonucleotides or ribonucleotides
`which do not have a nucleobase moicty. This kind of compoundis, among others, described
`in Sternbergeret al.(2002), Antisense. Nucl. Ac. Drug Dev.in press.
`
`Any of the aforementioned end modifications may be used in connection with the various
`
`embodiments of RNAi depicted in table 1. In connection therewith it is to be noted that any of
`the RNAi forms or embodiments disclosed herein with the sense strand being inactivated,
`preferably by having an end modification more preferably at the 5’ end, are particularly
`advantageous. This arises from the inactivation of the sense strand which corresponds to the
`
`13
`
`13
`
`

`

`a
`4m Saf
`Gust aie
`ot:
`AMM ees ab eR Lee ce
`
`
`14
`
`second strand of the ribonucleic acids described herein, which might otherwise interfere with
`an unrelated single-stranded RNA in the cell. Thus the expression and more particularly the
`translation pattern of the transcriptomeof a cell is more specifically influenced. This effectis
`also referred to as off-target effect. Referring to table 1
`those embodiments depicted as
`embodiments 7 and 8 are particularly adva